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Propionic acid (PPA) yog siv los kawm txog lub luag haujlwm ntawm mitochondrial dysfunction hauv neurodevelopmental disorders xws li autism spectrum disorder. PPA paub tias yuav cuam tshuam mitochondrial biogenesis, metabolism, thiab turnover. Txawm li cas los xij, cov teebmeem ntawm PPA ntawm mitochondrial dynamics, fission thiab fusion tseem muaj teeb meem vim yog qhov xwm txheej ntawm cov txheej txheem no. Ntawm no, peb siv cov txheej txheem quantitative imaging los tshawb nrhiav seb PPA cuam tshuam li cas rau mitochondrial ultrastructure, morphology, thiab dynamics hauv neuron-zoo li SH-SY5Y hlwb. PPA (5 mM) ua rau muaj kev txo qis tseem ceeb hauv thaj chaw mitochondrial (p < 0.01), Feret txoj kab uas hla thiab circumference (p < 0.05), thiab thaj chaw 2 (p < 0.01). Kev tshuaj xyuas qhov xwm txheej mitochondrial qhia tau tias muaj kev nce ntxiv tseem ceeb (p < 0.05) hauv fission thiab fusion xwm txheej, yog li tswj kev ncaj ncees ntawm mitochondrial network nyob rau hauv cov xwm txheej kev ntxhov siab. Ntxiv rau, qhov kev qhia tawm mRNA ntawm cMYC (p < 0.0001), NRF1 (p < 0.01), TFAM (p < 0.05), STOML2 (p < 0.0001) thiab OPA1 (p < 0.05) tau txo qis heev. 01). Qhov no qhia txog kev hloov kho dua tshiab ntawm mitochondrial morphology, biogenesis thiab dynamics kom tswj tau kev ua haujlwm nyob rau hauv cov xwm txheej kev ntxhov siab. Peb cov ntaub ntawv muab kev nkag siab tshiab rau hauv cov teebmeem ntawm PPA rau mitochondrial dynamics thiab qhia txog qhov siv tau ntawm cov txheej txheem duab rau kev kawm txog cov txheej txheem tswj hwm nyuaj uas koom nrog hauv kev teb rau kev ntxhov siab mitochondrial.
Mitochondria yog cov neeg koom nrog tseem ceeb hauv ntau yam haujlwm ntawm tes dhau ntawm lawv lub luag haujlwm ib txwm muaj hauv kev tsim hluav taws xob thiab biosynthesis. Mitochondrial metabolism yog tus tswj hwm tseem ceeb ntawm calcium signaling, metabolic thiab redox homeostasis, inflammatory signaling, epigenetic modifications, cell proliferation, differentiation thiab programmed cell death1. Tshwj xeeb, mitochondrial metabolism yog qhov tseem ceeb rau neuronal kev loj hlob, kev ciaj sia thiab kev ua haujlwm thiab yog dav cuam tshuam rau ntau yam kev tshwm sim ntawm neuropathology2,3,4.
Tau kaum xyoo dhau los no, cov xwm txheej metabolic tau tshwm sim ua tus tswj hwm tseem ceeb ntawm neurogenesis, kev sib txawv, kev loj hlob thiab kev hloov pauv 5,6. Tsis ntev los no, mitochondrial morphology thiab dynamics tau dhau los ua cov khoom tseem ceeb ntawm mitosis, ib qho txheej txheem dynamic uas tswj hwm lub pas dej ntawm mitochondria noj qab haus huv hauv cov hlwb. Mitochondrial dynamics raug tswj hwm los ntawm cov kev sib txuas ua ke xws li mitochondrial biogenesis thiab bioenergetics mus rau mitochondrial fission, fusion, transport thiab clearance7,8. Kev cuam tshuam ntawm ib qho ntawm cov txheej txheem integrative no ua rau kev tswj hwm ntawm cov tes hauj lwm mitochondrial noj qab haus huv thiab muaj cov txiaj ntsig zoo rau kev loj hlob ntawm lub hlwb9,10. Tseeb tiag, dysregulation ntawm mitochondrial dynamics tau pom nyob rau hauv ntau yam kev puas siab puas ntsws, neurodegenerative thiab neurodevelopmental kab mob, suav nrog autism spectrum disorders (ASD)11,12.
ASD yog ib qho kev puas siab puas ntsws ntawm lub hlwb uas muaj ntau yam kev hloov pauv ntawm cov noob caj noob ces thiab epigenetic. Qhov kev hloov pauv ntawm ASD tsis muaj kev sib cav, tab sis qhov tseem ceeb ntawm cov etiology molecular tseem tsis tau nkag siab zoo. Kev sau cov ntaub ntawv los ntawm cov qauv preclinical, kev tshawb fawb hauv tsev kho mob, thiab cov ntaub ntawv molecular ntau-omics muab cov pov thawj ntxiv ntawm kev ua haujlwm tsis zoo ntawm mitochondrial hauv ASD13,14. Yav dhau los peb tau ua qhov kev tshuaj ntsuam DNA methylation thoob plaws genome hauv ib pawg neeg mob uas muaj ASD thiab tau txheeb xyuas cov noob methylated sib txawv uas sib sau ua ke raws li mitochondrial metabolic pathways15. Tom qab ntawd peb tau tshaj tawm txog kev sib txawv ntawm cov regulators hauv nruab nrab ntawm mitochondrial biogenesis thiab dynamics, uas cuam tshuam nrog kev nce ntxiv ntawm mtDNA copy number thiab hloov pauv cov zis metabolic profile hauv ASD16. Peb cov ntaub ntawv muab cov pov thawj ntxiv tias mitochondrial dynamics thiab homeostasis ua lub luag haujlwm tseem ceeb hauv pathophysiology ntawm ASD. Yog li ntawd, kev txhim kho kev nkag siab txog kev sib raug zoo ntawm mitochondrial dynamics, morphology, thiab kev ua haujlwm yog lub hom phiaj tseem ceeb ntawm kev tshawb fawb txuas ntxiv mus rau hauv cov kab mob neurological uas muaj cov yam ntxwv ntawm kev ua haujlwm tsis zoo ntawm mitochondrial theem nrab.
Cov txheej txheem molecular feem ntau siv los kawm txog lub luag haujlwm ntawm cov noob caj noob ces tshwj xeeb hauv cov lus teb rau kev ntxhov siab mitochondrial. Txawm li cas los xij, txoj hauv kev no yuav raug txwv los ntawm ntau yam thiab lub sijhawm ntawm cov txheej txheem tswj mitotic. Ntxiv mus, kev qhia sib txawv ntawm cov noob caj noob ces mitochondrial yog qhov qhia tsis ncaj qha ntawm kev hloov pauv kev ua haujlwm, tshwj xeeb tshaj yog vim tsuas yog muaj tsawg tus noob caj noob ces feem ntau raug tshuaj xyuas. Yog li ntawd, ntau txoj hauv kev ncaj qha rau kev kawm txog kev ua haujlwm mitochondrial thiab bioenergetics tau raug pom zoo17. Mitochondrial morphology muaj feem cuam tshuam nrog mitochondrial dynamics. Mitochondrial duab, kev sib txuas, thiab cov qauv yog qhov tseem ceeb rau kev tsim hluav taws xob thiab mitochondrial thiab cell ciaj sia5,18. Ntxiv mus, ntau yam khoom ntawm mitosis tsom mus rau kev hloov pauv hauv mitochondrial morphology, uas yuav ua haujlwm ua qhov kawg ntawm mitochondrial dysfunction thiab muab lub hauv paus rau kev tshawb fawb tom qab.
Kev siv lub tshuab kuaj hluav taws xob (TEM) los saib xyuas cov qauv ntawm cov hlwb (mitochondrial morphology) tau yooj yim, thiab ua rau pom tau cov qauv ntawm cov hlwb no tau zoo dua. TEM pom tau cov qauv ntawm cov hlwb no, cov duab thiab cov qauv ntawm cov hlwb mitochondrial cristae thaum lub sij hawm uas cov hlwb mitochondrial nyob hauv lawv lub cev, tsis yog tsuas yog siv cov noob caj noob ces (gene transcription), cov protein expression lossis cov mitochondrial functional parameters hauv cov hlwb cell xwb.17,19,20. Tsis tas li ntawd xwb, TEM pab txhawb kev kawm txog kev sib cuam tshuam ntawm cov hlwb mitochondrial thiab lwm cov organelles, xws li endoplasmic reticulum thiab autophagosomes, uas ua lub luag haujlwm tseem ceeb hauv kev ua haujlwm ntawm cov hlwb mitochondrial thiab homeostasis21,22. Yog li, qhov no ua rau TEM yog qhov pib zoo rau kev kawm txog kev ua haujlwm tsis zoo ntawm cov hlwb mitochondrial ua ntej tsom mus rau cov txoj kev lossis cov noob caj noob ces tshwj xeeb. Thaum kev ua haujlwm ntawm cov hlwb mitochondrial ua rau muaj feem cuam tshuam rau neuropathology, muaj qhov xav tau kom kawm tau cov qauv ntawm cov hlwb mitochondrial thiab cov dynamics hauv cov qauv neuronal hauv vitro.
Hauv tsab xov xwm no, peb tshuaj xyuas cov mitochondrial dynamics hauv tus qauv neuronal ntawm mitochondrial dysfunction hauv autism spectrum disorder. Peb tau tshaj tawm txog kev sib txawv methylation ntawm propionyl-CoA carboxylase beta (PCCB) hauv ASD15, ib qho subunit ntawm mitochondrial propionyl-CoA carboxylase enzyme PCC. Kev tsis ua haujlwm ntawm PCC paub tias ua rau muaj kev sib sau ua ke ntawm cov propionyl derivatives, suav nrog propionic acid (PPA)23,24,25. PPA tau pom tias cuam tshuam rau neuronal metabolism thiab hloov tus cwj pwm hauv vivo thiab yog tus qauv tsiaj tsim rau kev kawm txog cov txheej txheem neurodevelopmental koom nrog ASD26,27,28. Tsis tas li ntawd, PPA tau tshaj tawm tias cuam tshuam rau mitochondrial membrane potential, biogenesis thiab respiration hauv vitro thiab tau siv dav los ua qauv mitochondrial dysfunction hauv neurons29,30. Txawm li cas los xij, qhov cuam tshuam ntawm PPA-induced mitochondrial dysfunction ntawm mitochondrial morphology thiab dynamics tseem tsis tau nkag siab zoo.
Txoj kev tshawb fawb no siv cov txheej txheem thaij duab sib txuas los ntsuas qhov cuam tshuam ntawm PPA rau mitochondrial morphology, dynamics, thiab kev ua haujlwm hauv SH-SY5Y hlwb. Ua ntej, peb tau tsim ib txoj kev TEM los pom kev hloov pauv hauv mitochondrial morphology thiab ultrastructure17,31,32. Muab qhov xwm txheej dynamic ntawm mitochondrial33, peb kuj tau siv mitochondrial event localizer (MEL) kev tshuaj xyuas los ntsuas qhov kev hloov pauv hauv qhov sib npaug ntawm fission thiab fusion xwm txheej, mitochondrial tus lej thiab ntim hauv qab PPA kev ntxhov siab. Thaum kawg, peb tau tshuaj xyuas seb mitochondrial morphology thiab dynamics puas cuam tshuam nrog kev hloov pauv hauv kev qhia tawm ntawm cov noob caj noob ces koom nrog biogenesis, fission, thiab fusion. Thaum ua ke, peb cov ntaub ntawv qhia txog qhov kev sib tw ntawm kev piav qhia qhov nyuaj ntawm cov txheej txheem tswj hwm mitochondrial dynamics. Peb hais txog qhov siv tau ntawm TEM hauv kev kawm mitochondrial morphology ua qhov ntsuas tau convergent endpoint ntawm mitosis hauv SH-SY5Y hlwb. Tsis tas li ntawd, peb hais tias TEM cov ntaub ntawv muab cov ntaub ntawv nplua nuj tshaj plaws thaum ua ke nrog cov txheej txheem thaij duab uas kuj ntes cov xwm txheej dynamic teb rau kev ntxhov siab metabolic. Kev piav qhia ntxiv txog cov txheej txheem tswj hwm molecular uas txhawb nqa neuronal cell mitosis yuav muab kev nkag siab tseem ceeb rau hauv mitochondrial Cheebtsam ntawm lub paj hlwb thiab cov kab mob neurodegenerative.
Txhawm rau ua rau muaj kev ntxhov siab ntawm mitochondrial, cov hlwb SH-SY5Y tau kho nrog PPA siv 3 mM thiab 5 mM sodium propionate (NaP). Ua ntej TEM, cov qauv tau raug rau kev npaj cov qauv cryogenic siv kev khov thiab khov siab (Daim Duab 1a). Peb tau tsim cov kav dej tshuaj xyuas duab mitochondrial tsis siv neeg los ntsuas yim qhov kev hloov pauv ntawm cov pej xeem mitochondrial thoob plaws peb qhov kev rov ua dua ntawm cov tsiaj txhu. Peb pom tias kev kho mob PPA tau hloov pauv plaub qhov kev hloov pauv: thaj tsam 2, thaj tsam, perimeter, thiab Feret txoj kab uas hla (Daim Duab 1b-e). Thaj tsam 2 tau txo qis nrog ob qho tib si 3 mM thiab 5 mM PPA kev kho mob (p = 0.0183 thiab p = 0.002, feem) (Daim Duab 1b), thaum thaj tsam (p = 0.003), perimeter (p = 0.0106) thiab Feret txoj kab uas hla tau txo qis tag nrho. Muaj kev txo qis tseem ceeb (p = 0.0172) hauv pawg kho mob 5 mM piv rau pawg tswj (Daim Duab 1c-e). Kev txo qis tseem ceeb hauv thaj chaw thiab qhov ncig qhia tau tias cov hlwb kho nrog 5 mM PPA muaj cov mitochondria me dua, ntau dua, thiab cov mitochondria no tsis ntev dua li cov hauv cov hlwb tswj. Qhov no kuj sib xws nrog kev txo qis tseem ceeb hauv Feret txoj kab uas hla, ib qho kev ywj pheej qhia txog kev txo qis ntawm qhov deb tshaj plaws ntawm cov npoo ntawm cov khoom me me. Kev hloov pauv hauv ultrastructure ntawm cristae tau pom: cristae tau dhau los ua tsawg dua nyob rau hauv kev cuam tshuam ntawm PPA kev ntxhov siab (Daim duab 1a, vaj huam sib luag B). Txawm li cas los xij, tsis yog txhua daim duab qhia meej txog ultrastructure ntawm cristae, yog li kev tshuaj xyuas ntau ntawm cov kev hloov pauv no tsis tau ua tiav. Cov ntaub ntawv TEM no yuav cuam tshuam peb qhov xwm txheej ua tau: (1) PPA txhim kho fission lossis inhibits fusion, ua rau mitochondria uas twb muaj lawm kom me me; (2) txhim kho biogenesis tsim cov mitochondria tshiab, me dua lossis (3) ua rau ob qho tib si mechanisms tib lub sijhawm. Txawm hais tias cov xwm txheej no tsis tuaj yeem sib txawv los ntawm TEM, kev hloov pauv morphological tseem ceeb qhia txog kev hloov pauv hauv mitochondrial homeostasis thiab dynamics nyob rau hauv PPA kev ntxhov siab. Tom qab ntawd peb tau tshawb nrhiav cov kev ntsuas ntxiv los piav qhia ntxiv txog cov dynamics no thiab cov mechanisms muaj peev xwm hauv qab lawv.
Propionic acid (PPA) hloov kho cov qauv mitochondrial. (a) Cov duab sawv cev ntawm kev xa hluav taws xob microscopy (TEM) qhia tias qhov loj me ntawm mitochondrial txo qis thiab mitochondria ua me dua thiab puag ncig dua nrog kev kho PPA ntau ntxiv; 0 mM (tsis kho), 3 mM thiab 5 mM, raws li. Cov xub liab qhia txog mitochondria. (b-e) SH-SY5Y hlwb kho nrog PPA rau 24 teev tau npaj rau TEM thiab cov txiaj ntsig tau soj ntsuam siv Fiji / ImageJ. Plaub ntawm yim qhov ntsuas tau qhia txog qhov sib txawv tseem ceeb ntawm kev tswj (tsis kho, 0 mM PPA) thiab kho (3 mM thiab 5 mM PPA) hlwb. (b) Cheeb Tsam 2, (c) Cheeb Tsam, (d) Perimeter, (e) Feret txoj kab uas hla. Kev tshuaj xyuas ib txoj kev ntawm qhov sib txawv (kev tswj vs. kev kho) thiab Dunnett qhov kev sib piv ntau yam tau siv los txiav txim siab qhov sib txawv tseem ceeb (p < 0.05). Cov ntsiab lus ntaub ntawv sawv cev rau qhov nruab nrab ntawm mitochondrial rau txhua lub cell, thiab cov kab yuam kev sawv cev rau qhov nruab nrab ± SEM. Cov ntaub ntawv qhia sawv cev rau n = 3, tsawg kawg yog 24 lub hlwb ib zaug rov ua dua; tag nrho ntawm 266 daim duab tau raug tshuaj xyuas; * qhia p < 0.05, ** qhia p < 0.01.
Yuav kom paub ntxiv txog seb mitochondrial dynamics teb li cas rau PPA, peb tau pleev xim rau mitochondria nrog tetramethylrhodamine ethyl ester (TMRE) thiab siv lub tshuab tsom xam lub sijhawm thiab MEL los ntsuas thiab ntsuas mitochondria tom qab 24 teev ntawm 3 thiab 5 mM PPA. Kev kho mob ntawm fission thiab fusion xwm txheej. (Daim duab 2a). Tom qab MEL kev tshuaj xyuas, mitochondria tau raug tshuaj xyuas ntxiv los ntsuas tus lej ntawm cov qauv mitochondrial thiab lawv qhov nruab nrab ntim. Peb pom muaj kev nce me me tab sis tseem ceeb ntawm tus lej ntawm cov xwm txheej fission tshwm sim ntawm 3 mM [4.9 ± 0.3 (p < 0.05)] piv rau fission [5.6 ± 0.3 (p < 0.05))] thiab fusion [5.4 ± 0.5 (p < 0.05)] thiab fusion [5.4 ± 0.5 (p < 0.05)] 0.05)] <0.05)] cov xwm txheej tau nce ntxiv ntawm 5 mM piv rau kev tswj hwm (Daim duab 3b). Tus lej ntawm mitochondria tau nce ntxiv ntawm ob qho tib si 3 [32.6 ± 2.1 (p < 0.05)] thiab 5 mM [34.1 ± 2.2 (p < 0.05)] (Daim duab 3c), thaum qhov nruab nrab ntawm txhua tus qauv mitochondrial tseem tsis hloov pauv (Daim duab 3c). 3d). Thaum muab ua ke, qhov no qhia tau hais tias kev kho dua tshiab ntawm mitochondrial dynamics ua haujlwm ua qhov kev teb rov qab uas ua tiav kev tswj hwm kev ncaj ncees ntawm lub network mitochondrial. Qhov nce ntawm tus lej ntawm cov xwm txheej fission ntawm 3 mM PPA qhia tau hais tias qhov nce ntawm tus lej mitochondrial yog ib feem vim yog mitochondrial fission, tab sis muab qhov nruab nrab ntawm mitochondrial ntim tseem tsis hloov pauv, biogenesis tsis tuaj yeem raug txiav txim siab ua qhov kev teb rov qab ntxiv. Txawm li cas los xij, cov ntaub ntawv no sib xws nrog cov qauv me me, puag ncig mitochondrial pom los ntawm TEM thiab kuj qhia txog kev hloov pauv tseem ceeb hauv mitochondrial dynamics uas tshwm sim los ntawm PPA.
Propionic acid (PPA) ua rau muaj kev hloov pauv mitochondrial kom tswj tau kev ncaj ncees ntawm lub network. Cov hlwb SH-SY5Y tau cog qoob loo, kho nrog 3 thiab 5 mM PPA rau 24 teev thiab pleev xim nrog TMRE thiab Hoechst 33342 ua raws li MEL kev tshuaj xyuas. (a) Cov duab sawv cev ntawm lub sijhawm-lapse microscopy uas qhia txog xim thiab binarized qhov siab tshaj plaws qhov kev kwv yees ntawm lub sijhawm 2 (t2) rau txhua qhov xwm txheej. Cov cheeb tsam xaiv tau qhia hauv txhua daim duab binary tau txhim kho thiab tso tawm hauv 3D ntawm peb lub sijhawm sib txawv (t1-t3) los qhia txog kev hloov pauv dhau sijhawm; cov xwm txheej fusion tau hais txog ntsuab; cov xwm txheej fission tau hais txog ntsuab. Tso tawm hauv liab. (b) Tus lej nruab nrab ntawm cov xwm txheej dynamic ib qho xwm txheej. (c) Tus lej nruab nrab ntawm cov qauv mitochondrial ib lub cell. (d) Qhov nruab nrab ntim (µm3) ntawm txhua tus qauv mitochondrial ib lub cell. Cov ntaub ntawv qhia yog sawv cev ntawm n = 15 lub hlwb ib pawg kho mob. Cov kab yuam kev qhia sawv cev rau qhov nruab nrab ± SEM, qhov ntsuas bar = 10 μm, * p < 0.05.
Propionic acid (PPA) ua rau muaj kev txwv tsis pub sau cov noob caj noob ces uas cuam tshuam nrog mitochondrial dynamics. Cov hlwb SH-SY5Y tau kho nrog 3 thiab 5 mM PPA rau 24 teev. Kev ntsuas noob caj noob ces tau ua tiav siv RT-qPCR thiab normalized rau B2M. Cov noob caj noob ces mitochondrial biogenesis (a) cMYC, (b) TFAM, (c) NRF1 thiab (d) NFE2L2. Cov noob caj noob ces mitochondrial fusion thiab fission (e) STOML2, (f) OPA1, (g) MFN1, (h) MFN2 thiab (i) DRP1. Qhov sib txawv tseem ceeb (p < 0.05) tau sim siv ib txoj kev ANOVA (tswj vs. kev kho) thiab Dunnett qhov kev sib piv ntau yam: * qhia p < 0.05, ** qhia p < 0.01, thiab **** qhia p < 0.0001. Cov kab sawv cev rau qhov kev qhia nruab nrab ± SEM. Cov ntaub ntawv qhia sawv cev rau n = 3 (STOML2, OPA1, TFAM), n = 4 (cMYC, NRF1, NFE2L2), thiab n = 5 (MFN1, MFN2, DRP1) cov kab mob sib kis.
Cov ntaub ntawv los ntawm TEM thiab MEL kev tshuaj xyuas ua ke qhia tau tias PPA hloov pauv mitochondrial morphology thiab dynamics. Txawm li cas los xij, cov txheej txheem imaging no tsis muab kev nkag siab rau hauv qab mechanisms tsav cov txheej txheem no. Yog li ntawd peb tau tshuaj xyuas qhov kev qhia mRNA ntawm cuaj tus neeg tswj hwm tseem ceeb ntawm mitochondrial dynamics, biogenesis, thiab mitosis teb rau PPA kev kho mob. Peb ntsuas cell myeloma oncogene (cMYC), nuclear respiratory factor (NRF1), mitochondrial transcription factor 1 (TFAM), NFE2-zoo li transcription factor BZIP (NFE2L2), gastrin-zoo li protein 2 (STOML2), optic nerve atrophy 1 (OPA1), Mitofusin 1 (MFN1), Mitofusin 2 (MFN2) thiab dynamin-related protein 1 (DRP1) tom qab 24 teev ntawm kev kho mob nrog 3 mM thiab 5 mM PPA. Peb tau pom 3 mM (p = 0.0053, p = 0.0415 thiab p < 0.0001, feem) thiab 5 mM (p = 0.0031, p = 0.0233, p < 0.0001) PPA kev kho mob. (Daim duab 3a-c). Qhov txo qis hauv kev qhia mRNA yog nyob ntawm koob tshuaj: qhov kev qhia ntawm cMYC, NRF1 thiab TFAM txo qis los ntawm 5.7, 2.6 thiab 1.9 zaug ntawm 3 mM, feem, thiab los ntawm 11.2, 3 thiab 2.2 zaug ntawm 5 mM. Qhov sib piv, lub hauv paus redox biogenesis gene NFE2L2 tsis tau hloov pauv ntawm txhua qhov kev sib sau ua ke ntawm PPA, txawm hais tias muaj qhov sib xws ntawm kev txo qis ntawm kev qhia tau pom (Daim duab 3d).
Peb kuj tau tshuaj xyuas qhov kev qhia tawm ntawm cov noob caj noob ces uas koom nrog kev tswj hwm ntawm fission thiab fusion. STOML2 xav tias koom nrog fusion, mitophagy thiab biogenesis, thiab nws qhov kev qhia tawm tau txo qis heev (p < 0.0001) los ntawm 3 mM (2.4-fold hloov) thiab 5 mM (2.8-fold hloov) PPA (Daim duab 1). 3d). Ib yam li ntawd, OPA1 fusion gene kev qhia tawm tau txo qis ntawm 3 mM (1.6-fold hloov) thiab 5 mM (1.9-fold hloov) PPA (p = 0.006 thiab p = 0.0024, feem) (Daim duab 3f). Txawm li cas los xij, peb tsis pom qhov sib txawv tseem ceeb hauv kev qhia tawm ntawm fusion genes MFN1, MFN2 lossis fission gene DRP1 nyob rau hauv 24-h PPA kev ntxhov siab (Daim duab 3g-i). Ntxiv rau, peb pom tias cov theem ntawm plaub cov protein fusion thiab fission (OPA1, MFN1, MFN2 thiab DRP1) tsis hloov pauv nyob rau hauv tib qho xwm txheej (Daim duab 4a-d). Nws yog ib qho tseem ceeb uas yuav tsum nco ntsoov tias cov ntaub ntawv no qhia txog ib qho taw tes hauv lub sijhawm thiab tej zaum yuav tsis qhia txog kev hloov pauv hauv kev qhia tawm protein lossis cov haujlwm thaum lub sijhawm thaum ntxov ntawm PPA kev ntxhov siab. Txawm li cas los xij, kev txo qis tseem ceeb hauv kev qhia tawm ntawm cMYC, NRF1, TFAM, STOML2, thiab OPA1 qhia txog kev hloov pauv tseem ceeb ntawm kev hloov pauv ntawm mitochondrial metabolism, biogenesis, thiab dynamics. Ntxiv rau, cov ntaub ntawv no qhia txog kev siv cov txheej txheem imaging los kawm ncaj qha txog kev hloov pauv kawg hauv mitochondrial function.
Cov theem protein fusion thiab fission factor tsis hloov pauv tom qab kev kho mob propionic acid (PPA). Cov hlwb SH-SY5Y tau kho nrog 3 thiab 5 mM PPA rau 24 teev. Cov theem protein tau ntsuas los ntawm kev tshuaj xyuas Western blot, thiab cov theem kev qhia tau normalized rau tag nrho cov protein. Kev qhia protein nruab nrab thiab cov sawv cev Western blots ntawm lub hom phiaj thiab tag nrho cov protein tau pom. a - OPA1, b - MFN1, c - MFN2, d - DRP1. Cov kab sawv cev rau qhov nruab nrab ± SEM, thiab cov ntaub ntawv tau pom yog sawv cev ntawm n = 3 biological replicates. Kev sib piv ntau yam (p < 0.05) tau ua tiav siv kev tshuaj xyuas ib txoj kev ntawm qhov sib txawv thiab Dunnett qhov kev xeem. Cov gel thiab blot thawj tau pom hauv Daim Duab S1.
Kev ua haujlwm tsis zoo ntawm Mitochondrial yog txuam nrog ntau yam kab mob xws li cov kab mob metabolic, kab mob plawv thiab cov leeg nqaij mus rau cov kab mob neurological1,10. Ntau yam kab mob neurodegenerative thiab neurodegenerative yog txuam nrog mitochondrial dysfunction, qhia txog qhov tseem ceeb ntawm cov organelles no thoob plaws lub neej ntawm lub hlwb. Cov kab mob no suav nrog tus kab mob Parkinson, tus kab mob Alzheimer thiab ASD3,4,18. Txawm li cas los xij, kev nkag mus rau cov ntaub so ntswg hauv lub hlwb los kawm txog cov kab mob no yog qhov nyuaj, tshwj xeeb tshaj yog ntawm theem mechanistic, ua rau cov qauv cellular yog lwm txoj hauv kev tsim nyog. Hauv kev tshawb fawb no, peb siv lub cev qauv cellular siv PPA-kho SH-SY5Y hlwb los rov hais dua qhov ua haujlwm tsis zoo ntawm mitochondrial uas pom hauv cov kab mob neuronal, tshwj xeeb tshaj yog autism spectrum disorders. Kev siv tus qauv PPA no los kawm txog mitochondrial dynamics hauv neurons yuav muab kev nkag siab rau hauv etiology ntawm ASD.
Peb tau tshawb nrhiav qhov ua tau ntawm kev siv TEM los saib cov kev hloov pauv hauv mitochondrial morphology. Nws yog ib qho tseem ceeb uas yuav tsum nco ntsoov tias TEM yuav tsum tau siv kom raug kom ua kom nws muaj txiaj ntsig zoo tshaj plaws. Kev npaj cov cryo-specimens tso cai rau kev khaws cia zoo dua ntawm cov qauv neuronal los ntawm kev kho cov khoom sib txuas ntawm tes thiab txo qhov kev tsim cov khoom cuav34. Sib xws nrog qhov no, peb tau pom tias cov hlwb neuron-zoo li SH-SY5Y muaj cov organelles subcellular thiab elongated mitochondria (Daim duab 1a). Qhov no qhia txog qhov siv tau ntawm cov txheej txheem cryogenic rau kev kawm mitochondrial morphology hauv cov qauv hlwb neuronal. Txawm hais tias kev ntsuas ntau yog qhov tseem ceeb rau kev tshuaj xyuas lub hom phiaj ntawm cov ntaub ntawv TEM, tseem tsis muaj kev pom zoo rau cov kev ntsuas tshwj xeeb uas yuav tsum tau ntsuas kom paub meej cov kev hloov pauv mitochondrial morphological. Raws li ntau tus neeg tshawb fawb uas tau tshuaj xyuas mitochondrial morphology17,31,32, peb tau tsim cov kav dej tshuaj xyuas duab mitochondrial uas ntsuas yim qhov kev ntsuas morphological, uas yog: thaj chaw, thaj chaw2, qhov sib piv, perimeter, circularity, degree, Feret txoj kab uas hla. thiab roundness.
Ntawm lawv, PPA txo qis thaj tsam 2, thaj tsam, perimeter, thiab Feret txoj kab uas hla (Daim duab 1b-e). Qhov no qhia tau hais tias mitochondria tau me dua thiab puag ncig dua, uas yog sib xws nrog cov kev tshawb fawb yav dhau los uas qhia txog kev txo qis hauv thaj chaw mitochondrial tom qab 72 teev ntawm PPA30-induced mitochondrial kev ntxhov siab. Cov yam ntxwv morphological no yuav qhia tau tias mitochondrial fission, ib qho txheej txheem tsim nyog los cais cov khoom puas tsuaj los ntawm lub network mitochondrial los txhawb lawv qhov kev puas tsuaj los ntawm mitophagy35,36,37. Ntawm qhov tod tes, qhov txo qis hauv qhov nruab nrab mitochondrial loj yuav cuam tshuam nrog kev nce biogenesis, uas ua rau muaj kev tsim cov mitochondria me me. Kev nce fission lossis biogenesis sawv cev rau kev teb rov qab los tswj mitosis tiv thaiv kev ntxhov siab mitochondrial. Txawm li cas los xij, kev loj hlob mitochondrial txo qis, kev sib xyaw tsis zoo, lossis lwm yam mob tsis tuaj yeem raug tshem tawm.
Txawm hais tias cov duab siab daws teeb meem uas tsim los ntawm TEM tso cai rau kev txiav txim siab ntawm cov yam ntxwv morphological ntawm qib ntawm tus kheej mitochondria, txoj kev no tsim cov duab thaij ob-seem ntawm ib qho taw tes hauv lub sijhawm. Txhawm rau kawm txog cov lus teb dynamic rau kev ntxhov siab metabolic, peb tau pleev xim rau mitochondria nrog TMRE thiab siv lub sijhawm-lapse microscopy nrog MEL tsom xam, uas tso cai rau kev pom 3D siab ntawm kev hloov pauv hauv lub network mitochondrial dhau sijhawm 33,38. Peb tau pom cov kev hloov pauv me me tab sis tseem ceeb hauv mitochondrial dynamics nyob rau hauv PPA kev ntxhov siab (Daim duab 2). Ntawm 3 mM, tus lej ntawm cov xwm txheej fission nce ntxiv ntau, thaum cov xwm txheej fusion tseem zoo ib yam li hauv kev tswj hwm. Kev nce ntxiv ntawm tus lej ntawm ob qho tib si fission thiab fusion xwm txheej tau pom ntawm 5 mM PPA, tab sis cov kev hloov pauv no yog kwv yees li proportional, qhia tias fission thiab fusion kinetics ncav cuag qhov sib npaug ntawm cov concentration siab dua (Daim duab 2b). Qhov nruab nrab mitochondrial ntim tseem tsis hloov pauv ntawm ob qho tib si 3 thiab 5 mM PPA, qhia tias kev ncaj ncees ntawm lub network mitochondrial tau khaws cia (Daim duab 2d). Qhov no qhia txog lub peev xwm ntawm cov tes hauj lwm mitochondrial dynamic los teb rau kev ntxhov siab me me kom tswj tau homeostasis yam tsis ua rau muaj kev sib cais ntawm tes hauj lwm. Ntawm 3 mM PPA, qhov nce ntawm fission txaus los txhawb kev hloov mus rau qhov sib npaug tshiab, tab sis xav tau kev kho dua tshiab kinetic ntau dua los teb rau kev ntxhov siab los ntawm cov concentration ntau dua ntawm PPA.
Tus naj npawb ntawm mitochondria nce ntxiv ntawm ob qho kev ntxhov siab PPA, tab sis qhov nruab nrab ntawm mitochondrial ntim tsis hloov pauv ntau (Daim Duab 2c). Qhov no yuav yog vim muaj biogenesis ntau ntxiv lossis kev faib ntau ntxiv; txawm li cas los xij, yog tias tsis muaj qhov txo qis tseem ceeb hauv qhov nruab nrab mitochondrial ntim, nws yuav muaj feem ntau dua uas biosynthesis nce ntxiv. Txawm li cas los xij, cov ntaub ntawv hauv Daim Duab 2 txhawb nqa qhov muaj ob txoj hauv kev them rov qab: kev nce ntxiv ntawm cov xwm txheej fission, sib xws nrog kev nce ntawm mitochondrial fission, thiab kev nce ntxiv ntawm cov xwm txheej, sib xws nrog mitochondrial biogenesis. Thaum kawg, kev them rov qab dynamic rau kev ntxhov siab me me yuav muaj cov txheej txheem sib xws uas cuam tshuam nrog fission, fusion, biogenesis, thiab mitophagy. Txawm hais tias cov kws sau ntawv yav dhau los tau qhia tias PPA txhim kho mitosis30,39 thiab mitophagy29, peb muab pov thawj rau kev kho dua tshiab ntawm mitochondrial fission thiab fusion dynamics teb rau PPA. Cov ntaub ntawv no lees paub qhov kev hloov pauv morphological pom los ntawm TEM thiab muab kev nkag siab ntxiv rau cov txheej txheem cuam tshuam nrog PPA-induced mitochondrial dysfunction.
Vim tias tsis yog TEM lossis MEL kev tshuaj xyuas tau muab pov thawj ncaj qha ntawm cov txheej txheem tswj hwm cov noob caj noob ces uas yog lub hauv paus ntawm kev hloov pauv morphological uas tau pom, peb tau tshuaj xyuas RNA kev qhia tawm ntawm cov noob caj noob ces uas koom nrog hauv mitochondrial metabolism, biogenesis, thiab dynamics. Lub cMYC proto-oncogene yog ib qho transcription factor uas koom nrog kev tswj hwm ntawm mitochondrial, glycolysis, amino acid thiab fatty acid metabolism40. Tsis tas li ntawd, cMYC paub tias tswj hwm kev qhia tawm ntawm yuav luag 600 mitochondrial noob caj noob ces uas koom nrog hauv mitochondrial transcription, kev txhais lus, thiab kev sib dhos ua ke, suav nrog NRF1 thiab TFAM41. NRF1 thiab TFAM yog ob lub hauv paus tswj hwm ntawm mitosis, ua haujlwm downstream ntawm PGC-1α los ua kom mtDNA replication. Txoj kev no tau ua haujlwm los ntawm cAMP thiab AMPK signaling thiab muaj kev nkag siab rau kev siv zog thiab kev ntxhov siab metabolic. Peb kuj tau tshuaj xyuas NFE2L2, tus tswj hwm redox ntawm mitochondrial biogenesis, los txiav txim siab seb cov teebmeem ntawm PPA puas yuav raug cuam tshuam los ntawm kev ntxhov siab oxidative.
Txawm hais tias NFE2L2 kev qhia tseem tsis tau hloov pauv, peb pom tias muaj kev txo qis ntawm cov tshuaj hauv kev qhia ntawm cMYC, NRF1 thiab TFAM tom qab 24 teev ntawm kev kho mob nrog 3 mM thiab 5 mM PPA (Daim duab 3a-c). Kev txo qis ntawm cMYC kev qhia tau raug tshaj tawm ua ntej tias yog kev teb rau kev ntxhov siab mitochondrial42, thiab qhov sib txawv, kev txo qis ntawm cMYC kev qhia tuaj yeem ua rau mitochondrial dysfunction los ntawm kev kho dua tshiab mitochondrial metabolism, kev sib txuas network, thiab membrane polarization43. Qhov nthuav yog, cMYC kuj koom nrog kev tswj hwm ntawm mitochondrial fission thiab fusion42,43 thiab paub tias yuav ua rau DRP1 phosphorylation thiab mitochondrial localization nce thaum lub sijhawm faib cell44, nrog rau kev sib tham txog mitochondrial morphological remodeling hauv neuronal stem cell45. Tseeb tiag, cMYC-deficient fibroblasts qhia txog kev txo qis mitochondrial loj, sib xws nrog kev hloov pauv los ntawm PPA43 kev ntxhov siab. Cov ntaub ntawv no qhia txog kev sib raug zoo tab sis tseem tsis tau meej meej ntawm cMYC thiab mitochondrial dynamics, muab lub hom phiaj nthuav rau kev kawm yav tom ntej ntawm PPA kev ntxhov siab ua rau muaj kev hloov kho dua tshiab.
Qhov kev txo qis ntawm NRF1 thiab TFAM yog sib xws nrog lub luag haujlwm ntawm cMYC ua tus neeg ua haujlwm tseem ceeb. Cov ntaub ntawv no kuj sib xws nrog cov kev tshawb fawb yav dhau los hauv tib neeg cov qog nqaij hlav qog nqaij hlav uas qhia tias PPA txo qis NRF1 mRNA kev qhia tawm ntawm 22 teev, uas tau cuam tshuam nrog ATP depletion thiab nce ROS46. Cov kws sau ntawv no kuj tau tshaj tawm tias TFAM kev qhia tawm nce ntawm 8.5 teev tab sis rov qab mus rau qib pib ntawm 22 teev. Hauv kev sib piv, Kim et al. (2019) tau qhia tias TFAM mRNA kev qhia tawm tau txo qis tom qab 4 teev ntawm PPA kev ntxhov siab hauv SH-SY5Y hlwb; txawm li cas los xij, tom qab 72 teev, TFAM protein kev qhia tawm tau nce siab thiab tus lej mtDNA luam tawm tau nce siab. Yog li, qhov kev txo qis ntawm cov noob caj noob ces mitochondrial biogenesis uas peb tau pom tom qab 24 teev tsis suav nrog qhov ua tau tias qhov kev nce ntxiv ntawm cov mitochondria yog cuam tshuam nrog kev ua haujlwm ntawm biogenesis ntawm cov sijhawm dhau los. Cov kev tshawb fawb yav dhau los tau qhia tias PPA ua rau PGC-1α mRNA thiab protein ntau dua hauv SH-SY5Y hlwb thaum 4 teev 30 feeb, thaum propionic acid ua rau mitochondrial biogenesis hauv calf hepatocytes ntawm PGC-1α thaum 12 teev 39 feeb. Qhov nthuav yog, PGC-1α tsis yog tsuas yog tus tswj kev sau ntawv ncaj qha ntawm NRF1 thiab TFAM, tab sis kuj tau pom tias tswj hwm kev ua haujlwm ntawm MFN2 thiab DRP1 los ntawm kev tswj hwm fission thiab fusion47. Thaum muab ua ke, qhov no qhia txog kev sib txuas ze ntawm cov txheej txheem tswj hwm mitochondrial compensatory teb uas tau tshwm sim los ntawm PPA. Ntxiv mus, peb cov ntaub ntawv qhia txog kev tsis sib haum xeeb ntawm kev tswj hwm kev sau ntawv ntawm biogenesis thiab metabolism hauv qab PPA kev ntxhov siab.
Cov noob STOML2, OPA1, MFN1, MFN2 thiab DRP1 yog cov tseem ceeb ntawm cov mitochondrial fission, fusion thiab dynamics37,48,49. Muaj ntau lwm cov noob koom nrog hauv mitochondrial dynamics, txawm li cas los xij, STOML2, OPA1 thiab MFN2 tau pom tias muaj methylated sib txawv hauv ASD cohorts,16 thiab ntau txoj kev tshawb fawb ywj pheej tau tshaj tawm cov kev hloov pauv hauv cov ntawv sau no los teb rau mitochondrial stress50,51.52. Kev qhia tawm ntawm ob qho tib si OPA1 thiab STOML2 tau txo qis los ntawm 3 mM thiab 5 mM PPA kev kho mob (Daim duab 3e, f). OPA1 yog ib qho ntawm cov regulators classical ntawm mitochondrial fusion los ntawm kev sib cuam tshuam ncaj qha nrog MFN1 thiab 2 thiab ua lub luag haujlwm hauv cristae remodeling thiab mitochondrial morphology53. Lub luag haujlwm tseeb ntawm STOML2 hauv mitochondrial dynamics tseem tsis meej, tab sis pov thawj qhia tias nws ua lub luag haujlwm hauv mitochondrial fusion, biogenesis, thiab mitophagy.
STOML2 koom nrog kev tswj hwm kev sib txuas ntawm mitochondrial respiratory thiab kev tsim cov pa respiratory chain complexes54,55 thiab tau pom tias hloov pauv cov yam ntxwv ntawm cov hlwb mob qog noj ntshav56. Cov kev tshawb fawb tau qhia tias STOML2 txhawb nqa mitochondrial membrane potential thiab biogenesis los ntawm kev sib cuam tshuam nrog BAN thiab cardiolipin55, 57, 58. Tsis tas li ntawd, cov kev tshawb fawb ywj pheej tau qhia tias kev sib cuam tshuam ntawm STOML2 thiab PINK1 tswj hwm mitophagy59,60. Qhov tseem ceeb, STOML2 tau tshaj tawm tias cuam tshuam ncaj qha nrog thiab ruaj khov MFN2 thiab kuj ua lub luag haujlwm tseem ceeb hauv kev ruaj khov OPA1 isoforms ntev los ntawm kev txwv tsis pub protease ua lub luag haujlwm rau OPA1 degradation53,61,62. Kev txo qis hauv STOML2 kev qhia pom hauv PPA reactions yuav ua rau cov protein fusion no yooj yim rau kev puas tsuaj los ntawm ubiquitin- thiab proteasome-dependent pathways48. Txawm hais tias lub luag haujlwm tseeb ntawm STOML2 thiab OPA1 hauv kev teb rov qab rau PPA tsis meej, kev txo qis ntawm cov noob fusion no (Daim Duab 3) yuav cuam tshuam qhov sib npaug ntawm fission thiab fusion thiab ua rau mitochondrial loj me me (Daim Duab 3). 1).
Ntawm qhov tod tes, OPA1 protein kev qhia tawm tseem tsis tau hloov pauv tom qab 24 teev, thaum cov mRNA thiab cov protein theem ntawm MFN1, MFN2 lossis DRP1 tsis hloov pauv ntau tom qab PPA kho (Daim duab 3g-i, Daim duab 4). Qhov no yuav qhia tau tias tsis muaj kev hloov pauv hauv kev tswj hwm ntawm cov yam no uas koom nrog mitochondrial fusion thiab fission. Txawm li cas los xij, nws tsim nyog sau cia tias txhua tus ntawm plaub lub noob no kuj tseem tswj hwm los ntawm kev hloov kho tom qab kev sau ntawv (PTMs) uas tswj cov protein ua haujlwm. OPA1 muaj yim qhov sib txawv splice variants uas yog proteolytically cleaved hauv mitochondria los tsim ob lub isoforms sib txawv 63. Qhov sib npaug ntawm cov isoforms ntev thiab luv luv thaum kawg txiav txim siab lub luag haujlwm ntawm OPA1 hauv mitochondrial fusion thiab kev saib xyuas ntawm mitochondrial network64. DRP1 kev ua haujlwm yog tswj hwm los ntawm calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylation, thaum DRP1 degradation yog tswj hwm los ntawm ubiquitination thiab SUMOylation65. Thaum kawg, ob qho tib si DRP1 thiab MFN1/2 yog GTPases, yog li kev ua ub no yuav raug cuam tshuam los ntawm tus nqi ntawm GTP tsim tawm hauv mitochondria 66. Yog li ntawd, txawm hais tias qhov kev qhia tawm ntawm cov protein no tseem nyob ruaj khov, qhov no yuav tsis qhia txog kev ua ub no ntawm cov protein lossis localization67,68 tsis hloov pauv. Tseeb tiag, cov protein repertoires PTM uas twb muaj lawm feem ntau ua haujlwm ua thawj kab ntawm kev tiv thaiv uas muaj lub luag haujlwm rau kev sib tham txog kev ntxhov siab sai. Thaum muaj kev ntxhov siab metabolic nruab nrab hauv peb tus qauv, nws zoo li PTM txhawb nqa kev ua ub no ntawm cov protein fusion thiab fission kom rov qab tau mitochondrial kev ncaj ncees yam tsis tas yuav tsum tau ua kom muaj zog ntxiv ntawm cov noob no ntawm mRNA lossis protein theem.
Thaum muab ua ke, cov ntaub ntawv saum toj no qhia txog qhov nyuaj thiab lub sijhawm nyob ntawm kev tswj hwm ntawm mitochondrial morphology thiab cov teeb meem ntawm kev piav qhia cov txheej txheem no. Txhawm rau kawm txog kev qhia txog cov noob caj noob ces, nws yog thawj qhov tsim nyog los txheeb xyuas cov noob caj noob ces tshwj xeeb hauv txoj kev. Txawm li cas los xij, peb cov ntaub ntawv qhia tias cov noob caj noob ces hauv tib txoj kev tsis teb rau tib txoj kev ntxhov siab. Qhov tseeb, cov kev tshawb fawb yav dhau los tau qhia tias cov noob caj noob ces sib txawv hauv tib txoj kev yuav ua rau pom cov qauv teb sib txawv ntawm lub sijhawm30,46. Tsis tas li ntawd, muaj cov txheej txheem tom qab sau ntawv nyuaj uas cuam tshuam kev sib raug zoo ntawm kev sau ntawv thiab kev ua haujlwm ntawm noob caj noob ces. Kev tshawb fawb Proteomic tuaj yeem muab kev nkag siab rau hauv qhov cuam tshuam ntawm PTMs thiab kev ua haujlwm ntawm cov protein, tab sis lawv kuj ua rau muaj kev cov nyom suav nrog cov txheej txheem qis-throughput, cov teeb liab siab-rau-suab nrov, thiab kev daws teeb meem tsis zoo.
Hauv cov ntsiab lus no, kev kawm txog mitochondrial morphology siv TEM thiab MEL muaj peev xwm zoo los teb cov lus nug tseem ceeb txog kev sib raug zoo ntawm mitochondrial dynamics thiab kev ua haujlwm thiab qhov no cuam tshuam li cas rau tus kab mob. Qhov tseem ceeb tshaj plaws, TEM muab ib txoj hauv kev ncaj qha rau kev ntsuas mitochondrial morphology ua qhov kawg ntawm mitochondrial dysfunction thiab dynamics51. MEL kuj muab ib txoj hauv kev ncaj qha rau kev pom fission thiab fusion cov xwm txheej hauv ib puag ncig cellular peb-dimensional, tso cai rau kev ntsuas ntawm dynamic mitochondrial remodeling txawm tias tsis muaj kev hloov pauv hauv gene expression33. Ntawm no peb hais txog qhov siv tau ntawm mitochondrial imaging techniques hauv cov kab mob mitochondrial theem nrab. Cov kab mob no feem ntau yog tus cwj pwm los ntawm kev ntxhov siab me me metabolic mob uas tus cwj pwm los ntawm kev hloov pauv me me ntawm cov tes hauj lwm mitochondrial es tsis yog kev puas tsuaj mitochondrial mob hnyav. Txawm li cas los xij, kev them nyiaj mitochondrial uas xav tau los tswj mitosis nyob rau hauv kev ntxhov siab ntev muaj cov txiaj ntsig zoo. Hauv cov ntsiab lus ntawm neuroscience, kev nkag siab zoo dua ntawm cov txheej txheem them nyiaj no yuav muab cov ntaub ntawv tseem ceeb txog pleiotropic neuropathology cuam tshuam nrog mitochondrial dysfunction.
Thaum kawg, peb cov ntaub ntawv qhia txog qhov siv tau ntawm cov txheej txheem duab rau kev nkag siab txog cov txiaj ntsig ntawm kev sib cuam tshuam ntawm cov noob caj noob ces, kev hloov pauv protein, thiab cov protein ua haujlwm uas tswj cov neuronal mitochondrial dynamics. Peb siv PPA los ua qauv mitochondrial dysfunction hauv tus qauv neuronal cell kom nkag siab txog mitochondrial Cheebtsam ntawm ASD. SH-SY5Y hlwb kho nrog PPA tau qhia txog kev hloov pauv hauv mitochondrial morphology: mitochondria tau dhau los ua me me thiab puag ncig, thiab cristae tsis zoo txhais thaum pom los ntawm TEM. MEL kev tshuaj xyuas qhia tau hais tias cov kev hloov pauv no tshwm sim ua ke nrog kev nce ntxiv ntawm fission thiab fusion xwm txheej los tswj lub network mitochondrial teb rau kev ntxhov siab me me. Ntxiv mus, PPA cuam tshuam rau kev tswj hwm transcriptional ntawm mitochondrial metabolism thiab homeostasis. Peb tau txheeb xyuas cMYC, NRF1, TFAM, STOML2, thiab OPA1 ua cov tswj hwm mitochondrial tseem ceeb uas cuam tshuam los ntawm PPA kev ntxhov siab thiab tej zaum yuav ua lub luag haujlwm hauv kev sib tham PPA-induced kev hloov pauv hauv mitochondrial morphology thiab kev ua haujlwm. Yuav tsum muaj kev tshawb fawb yav tom ntej kom paub meej txog kev hloov pauv ntawm lub sijhawm uas PPA ua rau muaj kev qhia txog cov noob caj noob ces thiab cov protein ua haujlwm, kev hloov pauv hauv zos, thiab kev hloov pauv tom qab kev txhais lus. Peb cov ntaub ntawv qhia txog qhov nyuaj thiab kev sib koom ua ke ntawm cov txheej txheem tswj hwm uas cuam tshuam rau kev teb rau kev ntxhov siab ntawm mitochondrial thiab qhia txog kev siv TEM thiab lwm yam kev siv duab rau kev tshawb fawb txog cov txheej txheem ntau dua.
Cov kab mob SH-SY5Y cell (ECACC, 94030304-1VL) tau yuav los ntawm Sigma-Aldrich. Cov hlwb SH-SY5Y tau loj hlob hauv Dulbecco's modified Eagle's medium/F-12 nutrient mixture (DMEM/F-12) thiab L-glutamine (SC09411, ScienCell) hauv 25 cm2 flasks ntxiv nrog 20% ​​fetal bovine serum (FBS) (10493106, ThermoFisher Scientific) thiab 1% penicillin-streptomycin (P4333-20ML, Sigma-Aldrich) ntawm 37 °C, 5% CO2. Cov hlwb tau subcultured rau 80% confluence siv 0.05% trypsin-EDTA (15400054, ThermoFisher Scientific), centrifuged ntawm 300 g thiab plated ntawm qhov ceev ntawm kwv yees li 7 × 105 hlwb / ml. Tag nrho cov kev sim tau ua rau ntawm cov hlwb SH-SY5Y uas tsis sib txawv ntawm cov kab lus 19–22. PPA yog muab ua NaP. Yaj NaP hmoov (CAS No. 137-40-6, cov mis tshuaj C3H5NaO2, P5436-100G, Sigma-Aldrich) hauv dej sov MilliQ kom muaj qhov concentration ntawm 1 M thiab khaws cia ntawm 4 ° C. Nyob rau hnub kho mob, dilute cov tshuaj no nrog 1 M PPA rau 3 mM thiab 5 mM PPA hauv cov tshuaj tsis muaj serum (DMEM/F-12 nrog L-glutamine). Cov concentration kho mob rau txhua qhov kev sim tsis muaj PPA (0 mM, tswj), 3 mM, thiab 5 mM PPA. Cov kev sim tau ua tiav hauv tsawg kawg peb qhov kev rov ua dua ntawm cov kab mob.
Cov qe SH-SY5Y tau muab noob rau hauv 25 cm5 flasks ntawm tus nqi ntawm 5.5 × 105 qe/ml thiab loj hlob rau 24 teev. Kev kho mob PPA tau ntxiv rau hauv lub raj mis ua ntej 24 teev ntawm kev sib xyaw. Sau cov qe pellets ua raws li cov txheej txheem ib txwm ntawm cov tsiaj txhu (piav qhia saum toj no). Rov ua dua cov qe pellet hauv 100 µl 2.5% glutaraldehyde, 1 × PBS thiab khaws cia ntawm 4 ° C kom txog thaum ua tiav. Cov qe SH-SY5Y tau centrifuged luv luv kom pellet cov qe thiab tshem tawm 2.5% glutaraldehyde, 1 × PBS kua. Rov ua dua cov av noo hauv 4% agarose gel npaj rau hauv dej distilled (qhov piv ntawm agarose rau cov av noo yog 1: 1). Cov khoom Agarose tau muab tso rau ntawm cov phiaj ntawm cov phaj tiaj tus thiab coated nrog 1-hexadecene ua ntej khov siab. Cov qauv tau khov hauv 100% qhuav acetone ntawm -90 ° C rau 24 teev. Tom qab ntawd, qhov kub tau nce mus txog -80 ° C thiab cov tshuaj ntawm 1% osmium tetroxide thiab 0.1% glutaraldehyde tau ntxiv. Cov qauv tau khaws cia ntawm -80 ° C rau 24 teev. Tom qab qhov no, qhov kub tau maj mam nce mus rau qhov kub hauv chav tsev ntau hnub: ntawm - 80 ° C txog - 50 ° C rau 24 teev, txog - 30 ° C rau 24 teev, txog - 10 ° C rau 24 teev thiab thaum kawg mus rau qhov kub hauv chav tsev. kub.
Tom qab npaj cryogenic, cov qauv tau impregnated nrog resin thiab ultrathin seem (~ 100 nm) tau ua siv Leica Reichert UltracutS ultramicrotome (Leica Microsystems). Cov seem tau stained nrog 2% uranyl acetate thiab lead citrate. Cov qauv tau pom siv FEI Tecnai 20 transmission electron microscope (ThermoFisher (yav tas los hu ua FEI), Eindhoven, Lub Netherlands) ua haujlwm ntawm 200 kV (Lab6 transmitter) thiab lub koob yees duab Gatan CCD (Gatan, UK) nruab nrog Tridiem lub lim dej zog.
Hauv txhua qhov kev rov ua dua, tsawg kawg yog 24 daim duab ntawm tes tau txais, rau tag nrho 266 daim duab. Tag nrho cov duab tau raug tshuaj xyuas siv thaj tsam ntawm kev txaus siab (ROI) macro thiab Mitochondria macro. Lub mitochondrial macro yog raws li cov txheej txheem luam tawm 17,31,32 thiab tso cai rau kev ua tiav cov duab TEM semi-automated hauv Fiji / ImageJ69. Hauv nutshell: daim duab yog inverted thiab inverted siv dov pob keeb kwm yav dhau rho tawm (60 pixel radius) thiab FFT bandpass lim (siv 60 thiab 8 pixel sab saud thiab qis dua ciam teb) thiab kev txwv kab ntsug nrog kev kam rau siab ntawm 5%. Daim duab ua tiav tau txiav txim siab siv qhov siab tshaj plaws entropy algorithm thiab lub ntsej muag binary yog tsim. Cov cheeb tsam duab cuam tshuam nrog ROIs xaiv manually hauv cov duab TEM raw tau rho tawm, piav qhia txog mitochondria thiab tsis suav nrog cov plasma membrane thiab lwm thaj chaw siab-contrast. Rau txhua qhov ROI uas tau rho tawm, cov khoom me me binary loj dua 600 pixels tau raug tshuaj xyuas, thiab thaj chaw ntawm cov khoom me me, perimeter, cov axes loj thiab me, Feret txoj kab uas hla, qhov puag ncig, thiab qhov ncig tau ntsuas siv cov haujlwm ntsuas uas tau tsim los ntawm Fiji/ImageJ. Ua raws li Merrill, Flippo, thiab Strack (2017), thaj chaw 2, qhov sib piv ntawm cov khoom me me (qhov sib piv ntawm cov axis loj rau me), thiab cov duab zoo li cas (FF) tau suav los ntawm cov ntaub ntawv no, qhov twg FF = perimeter 2/4pi x thaj chaw. Cov lus txhais ntawm cov qauv parametric tuaj yeem pom hauv Merrill, Flippo, thiab Strack (2017). Cov macros uas tau hais muaj nyob rau ntawm GitHub (saib Cov Lus Qhia Txog Kev Muaj Cov Ntaub Ntawv). Qhov nruab nrab, kwv yees li 5,600 cov khoom me me tau raug tshuaj xyuas ib qho kev kho PPA, rau tag nrho kwv yees li 17,000 cov khoom me me (cov ntaub ntawv tsis tau qhia).
Cov hlwb SH-SH5Y tau muab tso rau hauv 8-chamber kab lis kev cai tais diav (ThermoFisher, #155411) kom tso cai rau kev sib txuas thaum hmo ntuj thiab tom qab ntawd incubated nrog TMRE 1: 1000 (ThermoFisher, #T669) thiab Hoechst 33342 1: 200 (Sigma-Aldrich, H6024). dyeing. Cov duab tau txais los ntawm kev siv 405 nm thiab 561 nm lasers dhau 10 feeb ib puag ncig, thiab cov duab raw tau txais los ua z-stacks uas muaj 10 daim duab micrographs nrog az kauj ruam ntawm 0.2 μm ntawm cov thav duab duab ntawm 12 lub sijhawm tom qab. Cov duab tau sau los ntawm kev siv Carl Zeiss LSM780 ELYRA PS.1 super-resolution platform (Carl Zeiss, Oberkochen, Lub Tebchaws Yelemees) siv LCI Plan Apochromate 100x / 1.4 Roj DIC M27 lens. Cov duab tau raug tshuaj xyuas hauv ImageJ siv cov kav dej uas tau piav qhia ua ntej thiab ImageJ plugin los ntsuas cov xwm txheej fusion thiab fission, tus lej nruab nrab ntawm cov qauv mitochondrial, thiab qhov nruab nrab ntawm mitochondrial ntim ib lub cell33. MEL macros muaj nyob rau ntawm GitHub (saib Cov Lus Qhia Txog Kev Muaj Cov Ntaub Ntawv).
Cov hlwb SH-SY5Y tau loj hlob hauv rau-qhov phaj ntawm qhov ceev ntawm 0.3 × 106 hlwb / mL rau 24 teev ua ntej kev kho mob. RNA tau rho tawm siv Quick-RNA ™ Miniprep protocol (ZR R1055, Zymo Research) nrog kev hloov kho me ntsis: ntxiv 300 μl ntawm RNA lysis buffer rau txhua qhov dej ua ntej tshem tawm thiab lyse txhua tus qauv ua kauj ruam kawg nrog 30 μl ntawm DNase / RNase elution. -free dej. Tag nrho cov qauv tau kuaj xyuas kom muaj nuj nqis thiab zoo siv NanoDrop ND-1000 UV-Vis Spectrophotometer. Tag nrho cov protein los ntawm cov hlwb lysates tau txais siv 200 μl RIPA lysis buffer, thiab cov protein concentration tau ntsuas siv Bradford protein assay70.
Kev tsim cov cDNA tau ua tiav los ntawm kev siv Tetro™ cDNA Synthesis Kit (BIO-65043, Meridian Bioscience) raws li cov lus qhia ntawm cov chaw tsim khoom nrog qee qhov kev hloov kho. cDNA tau tsim ua ke hauv 20-μl cov tshuaj tiv thaiv siv 0.7 txog 1 μg ntawm tag nrho RNA. Cov primers tau xaiv los ntawm cov ntawv luam tawm yav dhau los 42, 71, 72, 73, 74, 75, 76, 77, 78 (Rooj S1) thiab cov probes nrog tau tsim los ntawm kev siv cov cuab yeej PrimerQuest los ntawm Integrated DNA Technologies. Txhua cov noob caj noob ces uas xav tau tau ua kom zoo li qub rau lub noob caj noob ces nuclear B2M. Kev qhia noob caj noob ces ntawm STOML2, NRF1, NFE2L2, TFAM, cMYC thiab OPA1 tau ntsuas los ntawm RT-qPCR. Cov khoom sib xyaw ua ke suav nrog LUNA Taq polymerase (M3003L, New England Biolabs), 10 μM cov primers pem hauv ntej thiab rov qab, cDNA, thiab PCR-qib dej kom tau qhov ntim kawg ntawm 10 μL rau txhua qhov kev cuam tshuam. Kev qhia tawm ntawm cov noob faib thiab fission (DRP1, MFN1/2) tau ntsuas siv TaqMan multiplex assays. Luna Universal Probe qPCR Master Mix (M3004S, New England Biolabs) tau siv raws li cov lus qhia ntawm cov chaw tsim khoom nrog kev hloov kho me me. Cov khoom sib xyaw ua ke multiplex RT-qPCR suav nrog 1X LUNA Taq polymerase, 10 μM cov primers pem hauv ntej thiab rov qab, 10 μM probe, cDNA, thiab PCR-qib dej, ua rau qhov ntim kawg ntawm 20 μL rau txhua qhov kev cuam tshuam. RT-qPCR tau ua tiav siv Rotor-Gene Q 6-plex (QIAGEN RG—serial number: R0618110). Cov xwm txheej cycling tau qhia hauv Rooj S1. Tag nrho cov qauv cDNA tau raug nthuav dav hauv peb daim thiab ib daim nkhaus txheem tau tsim los ntawm kev siv cov txheej txheem ntawm kaum npaug dilutions. Cov Outliers hauv cov qauv peb daim nrog lub voj voog qhov sib txawv ntawm tus qauv (Ct)> 0.5 tau raug tshem tawm ntawm qhov kev tshuaj xyuas kom ntseeg tau tias cov ntaub ntawv rov ua dua 30,72. Kev qhia txog cov noob caj noob ces tau suav nrog siv txoj kev 2-ΔΔCt79.
Cov qauv protein (60 μg) tau sib xyaw nrog Laemmli loading buffer ntawm qhov sib piv 2: 1 thiab khiav ntawm 12% cov protein gel tsis muaj xim (Bio-Rad # 1610184). Cov protein tau hloov mus rau PVDF (polyvinylidene fluoride) daim nyias nyias (# 170-84156, Bio-Rad) siv lub tshuab Trans-Blot Turbo (# 170-4155, Bio-Rad). Daim nyias nyias tau thaiv thiab incubated nrog cov tshuaj tiv thaiv thawj zaug (OPA1, MFN1, MFN2, thiab DRP1) (diluted 1: 1000) rau 48 teev, ua raws li kev incubation nrog cov tshuaj tiv thaiv thib ob (1: 10,000) rau 1 teev. Cov daim nyias nyias tau raug thaij duab siv Clarity Western ECL Substrate (# 170-5061, Bio-Rad) thiab kaw cia siv lub tshuab Bio-Rad ChemiDoc MP. ImageLab version 6.1 tau siv rau kev tshuaj xyuas Western blot. Cov gel thiab blot thawj tau qhia nyob rau hauv Daim Duab S1. Cov ntaub ntawv txog Antibody tau muab nyob rau hauv Rooj S2.
Cov ntaub ntawv teeb tsa tau nthuav tawm ua qhov nruab nrab thiab qhov yuam kev txheem ntawm qhov nruab nrab (SEM) ntawm tsawg kawg peb qhov qauv ywj pheej. Cov ntaub ntawv teeb tsa tau sim rau qhov ib txwm siv qhov kev xeem Shapiro-Wilks (tshwj tsis yog hais lwm yam) ua ntej xav tias Gaussian faib thiab sib npaug ntawm cov qauv sib txawv thiab txuas ntxiv nrog kev tshuaj xyuas. Ntxiv nrog rau kev tshuaj xyuas cov ntaub ntawv teeb tsa siv Fisher's MEL LSD (p < 0.05), ib txoj kev ANOVA (kev kho mob vs. tswj nruab nrab), thiab Dunnett qhov kev sib piv ntau yam los txiav txim siab qhov tseem ceeb (p < 0.05). Cov nqi p tseem ceeb tau pom hauv daim duab ua *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Txhua qhov kev tshuaj xyuas lej thiab cov duab tau ua thiab tsim los ntawm kev siv GraphPad Prism 9.4.0.
Cov macro Fiji/ImageJ rau kev tshuaj xyuas duab TEM muaj rau pej xeem siv ntawm GitHub: https://github.com/caaja/TEMMitoMacro. Cov macro Mitochondrial Event Locator (MEL) muaj rau pej xeem siv ntawm GitHub: https://github.com/rensutheart/MEL-Fiji-Plugin.
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